International Conference, Tampa Bay. J.M.Lawrence, ed. (A.A.Balkema, Rotterdam, 1982) Echinoid metamorphosis: retraction and resorption of larval tissues

ROBERT D.BURKE University of Victoria, Canada

1 ABSTRACT Highsmith, 1977). It is not certain how larvae perceive these cues, though sensory During the first fifteen minutes of meta- receptors have been identified which may morphosis in punctulata and be involved in the perception of tactile Dendraster excentricus, larval tissues are components (Burke, 1980). There are several retracted and resorbed into the aboral accounts of the fates of larval and adult surface of the juvenile. Ultrastructural tissues at metamorphosis (MacBride, 1903; observations of the sequence indicate that Chia and Burke, 1978) and several of the there is an immediate contraction of the developmental processes of metamorphosis epidermis mediated by -like filaments. have been identified (Cameron and Hine- Within five minutes, the epidermis begins gardner, 1978; Chia and Burke, 1978). to histolysize and undergoes necrosis. The purpose of this report is to present Metamorphosis can be induced by brief, preliminary findings of studies of echinoid electrical stimulation of either of two metamorphosis in which the main objectives regions of neuropile. Catecholamine neuro- are to characterize developmental processes transmitters will initiate the contraction- of metamorphosis and determine how these histolysis-necrosis sequence in isolated processes are initiated and controlled. larval arms. Treatment with the ionophores This paper describes the sequence of devel- A23187 or X537A will induce only contrac- opmental events that take place in the tion, whereas histolysis without contrac- larval arms during metamorphosis. The arms tion can be induced by treatment with an are specialized larval structures which extract of histolysizing larval tissues. degenerate during metamorphosis. Results It is proposed that the nervous system are presented that suggest the retraction controls initiation of the retraction and and resorption of larval tissues may be resorption sequence during echinoid meta- controlled in part by the larval nervous morphosis. system.

2 INTRODUCTION 3 MATERIALS AND METHODS

It has been reported for several species of All larvae were cultured using the standard echinoids that certain environmental cues procedures outlined by Hinegardner (1969) will stimulate competent larvae to metamor- and Strathmann (1968). Adult Dendraster phose (Strathmann, 1978 for review). excentricus were collected intertidally at Presumably, the environmental cues act as Sidney Island, B.C. Canada, and adult stimuli for receptors on the larva. The Arbacia punctulata were collected sub- receptors, in turn, communicate with the tidally at St. Lucie Inlet, Fla. USA. tissues that are the effectors of metamor- Larvae were prepared for transmission phosis. The developmental processes that electron microscopy (TEM) by initially transform or activate these tissues are the fixing in a solution containing 2.5% glutar- response, metamorphosis. Evidence for such aldehyde, 0.2 M phosphate buffer, and 0.14 M a hypothesized stimulus-response mechanism NaCl. Specimens were post-fixed for 1 hr is not complete. Environmental cues have at room temperature in 2% osmium tetroxide been demonstrated but not fully character- in 1.25* sodium bicarbonate. Dehydration ized (Cameron and Hinegardner, 1974; was by alcohol exchange and specimens were

513 infiltrated and embedded in Epon. Sections skeletal rod piercing the ti] of the arm were stained with 50"; ethanol saturated and the tube of epidermis collapsing and with uranyl acetate and lead hydroxide folding as it is drawn towards the base of chelated with sodium citrate. Observations the arm. Th^ skeletal rod is eventually and micrographs were m .de using a Zeiss EM dropped from the aboral surface of the 9S-2. j uvcnile. Eor experiments with the ionophores The structural changes accompanying the A23187 and X537A (courtesy of W.E. Scott, retraction of the larval arms are first Hoffman-LaRoche Ltd.), and neurotransmitter apparent in tissues fixed thirty seconds substances, individual larval arms were after retraction begins. Bundles of micro- dissected from competent larvae with fine filaments (5 to 7 nm in diameter) which are tungsten needles and placed in test solu- not apparent in competent larvae occur in tions in depression slides or small watch the cvtoplasm of cells of the epidermis and glasses. The ionophores are not readily the ciliary bandr (Fig. 2). In the epi- soluable in sea water, so dimethyl sulf- dermal cells, the microfilarnent bundles oxide (DMSO) was used to nr»-pare stock occur throughout the cytoplasm and seem most solutions. Final test solutions contained numerous in the cells adjacent to the 2" DMSO. To test the effects of electrical ciliary bands (Fig. 3). The filaments are stimulation, competent larvae were attached usually oriented along the long axis of the to a fine tipped, polyethylene suction arm. Filaments within the ciliary bands electrode (tip diameter 40-60 ].im) and occur in both apical and basal portions of stimulated with either a D.C. square wave the ciliated cells. In the apical regions stimulator or a regulated D.C. source. of the ciliated cells the filaments are oriented at right angle to the long axis of the arm (Fig. 5), whereas in the basal 4 RESULTS portions of the cell the filaments are oriented along the long axis of the cells. 4. 1 rval arms In larvae fixed two minutes after the beginning of arm r^^act ion, the epidermis Competent echinoplutei have eight larval is pleated and folded as the arm contracts, arms which in the case of Arbacia punctu- and the ciliary bands fold inwards toward lata may be up to 800 um long. The arms the center of the arm. Filament bundles are outlined by a dense band of cilia and arc more numerous in the cells of the epi- function as locomotory and feeding organs dermis than a ter 30 seconds and are also throughout the life of the larva. In apparent within the mesenchymal cells Arbacia punctulata the arms are circular associated with the skeletal rod (Fig. 4). in cross section (Fig. It). The exterior The c_i 1 iary bands become disorganized as surface of the arm is comprised o: squarn- they to^d inward, fch6 cells detach and ous epithelium 1 to 2 pm thick and the begin 'o round ip (Fig. lb). Within the surface of the arm directec towards the cytoplusm of the eiHated cells axonemes larval mouth is columnar epithelium 4 urn and fibrils of cllii may be found (Fig. 6). thick. The ciliary bands are thickenings After five minutes the arms have retracted of the epidermis that are elliptical in to about one nuartor nf their original cross section and are comprised of spindle length. The , irms a^e now a thin cuboidal shaped cells each of which has a single epithelium surrroundi :ig a core of loosely cilium at its apical surface. A sinqLe associated individual cells (Fig. lc). The tract of axons, 1 to 2 urn in diameter lies c 11s within the core are the cells of the in a central position embedded in the base ciliary bands, tie axonal tracts, and the of the ciliary band. Each arm is supported mesenchyme. Fi_l aments can sti: 1 be observe^, by a single skeletal rod which is located within the covering epithelium and some of in the central space of the arms and has the cells within the core. However, ciliarv associated with it numerous mesencxymal band cells in the core of the retracted arms are rnnnded-up, have lost their apical-basal Metamorphosis begins with the eversion p^lirity, and have nuclei that are more of an aoulc rudiment from an inpoc'-:et ing granular and electron dense than at previous on the left side of the larva (see iiyman, stages (Fig. 7). Cytoplasmis debris is 1955 for review). The aversion is usually scattered throughout the core of the arms, completed in about 3 to 5 minutes. Begin- and the cells can be seen to have frag- ning at the same time as the eversion, the mented . larval cirrus are retracted into what will After ten minutes the larVal arms have become the abora1 surface of the juvenile. retracted to small knobs on the aboral sur- The retraction r icuires about 20 minutes iace. The cuboidal epithelium covering the to be completed. It begins with the arms has fewer filaments than at previous

514 Fig. 1. Licjht micrographs of cross sections of ] irval arms of7 Arbacia "mnctulata a) prior to metamorphosis, b) two minutes af tei the beuirning o" arm re; L'jrtio , c) Live minutes after the beginning of arm retraction. Kir - 1:) IJRI. Fig. 2. Transmission electron micrograph o:: epithelial cell of larval riri of Arbacia punctulata fixed thirty seconds a:tor t he ben^ning of arm rptraction Arrows indicate f iJ ,:m^nts. Bar - 0.5 um. Fig. 3. Longitudinal section through cells adjacent to the ciliary band in larval arm of Arbacia punctulata fixed thirty seconds after the beginning of arm retraction. Arrows indicate filaments. Bar - 0.5 pm. Fig. 4. Longitudinal section through portion of mesenchymal cel.l associated with larval skeleton in arm of Arbacia punctjL.ta fixed two minutes a:Lor the beginning o! arm retraction. Arrows indicate filaments. Bar - 0,5 \m. Fig. 5. Cross section of larval arm showing apical region of ciliated cells of ciliary band two minutes after the beginning of arm retraction. Arrows indicate filaments. Bar - 0.5 wm. Fig. 6. Basal region of ciliated cells of the ciliary band of larval arm of Arbacia punctulata fixed two minutes after the beginning of arm retraction. Note axonome and fibrils of retracted cilium. Bar - 0.5 pro. Fig. 7. Core region of retracted arm ten minutes after the beginning of arm retraction. Bar - 1 pm. Fig. 8. Core re..on of retracted arm of Arbacia punctulata twenty minutes after the beginning of am retraction. Large multinucleate mesenchymal cell shows evidence of phagocytotic activity. Bar - 1 ym. ' >..•

Fig. 9. Larval arm of Arbacia punctulata excised immediately after the beginning of arm retraction. a) 3 minutes after the beginning of ar retraction, b) 5 minutes, c) 10 minutes, contraction is completed, d) Fifteen minutes, note beginnings of histoiysis, e) 20 minutes, f) 25 minutes. Bar - 50 vim.

stages. The core is largely composed of Contraction draws both ends of the arms rounded cells usually containing the rem- towards the middle, presumably because nants of a cilium and a condensed, pyknotic neither end of the tube of epidermis is nucleus. Axonal tracts, although greatlv attached. After firteen minutes the arm disorganized can be identified within the is a ball of ce_ls pierced with the skele- core. As well, there are cells which appear tal rod. Beginning at about the time that to have been mesenchyme, identifiable bv the arm is fully contracted, the cells their ultrastructural appearance and their dissociate. After thirty minutes the arm multinucleate condition, scattered through- becomes a mass of individual cells. out the core. In specimens fixed 20 minutes Isolated larval arms treated with the after the beginning of arm retraction these ionophores A2J187 (2 X 10"\ 2 X 10"5) or multinucleate cells have begun phagooytizing X537A (5 ug/ml, 1.0 uq/ml) undergo contrac- the necrotic ciliated cells and the cyto- tion of the larva] epidermis 10 minutes plasmic debris of the core (Fig. ft) . after the beginning of treatment (Table 1). Arms rarely contracted to less than z/3 OI their original length and histoiysis does 4.2 Isolated larval arms not follow contraction. Treatment with 2% DMSO which is used as a solubiliziag Arms of larvae can be dissected from the body by cutting through the base of the arm with a fine tungsten needle. If an arm is Table 1. Summary of experiment with iono- removed from a competent larva not only phores using excised arms of Arbacia does the larva seem to be unaffected by its punctulata. Results arc 1rom 3 trials of absence, but the isolated arm is able to each treatment, 3 arms r>er trial. swim by means of its cilia for periods of up to 48 hours after it has been removed. 2X10-Y-4 4 M 10 ug/ml DMSO Isolated arms fixed one hour after excision A 23187 X 5 17A are ultrastructurally indistinguishable from arms that have not been removed. Reversibility rover- not re- no Arms removed from metamorphosing larvae (after 15 min) sible versible effect that have just begun the retraction of the larval epidermis will complete the entire 10 2M EDTA no con- no con- no sequence of contraction and histoiysis that traction traction effect occurs in arms that have not been excised (Fig. 9). Ultrastructural characteristics 10 lanthanum no con- no con- no of the sequence in excised arms are ident- nitrate traction traction effect ical to those described for intact larvae. Fig. 10. A competent larva of Dendraster excentricus attached by its apical neural plexus to a suction electrode, a) prior to stimulation, b) 30 seconds after stimulation, the adult rudiment is beginning to evert, c) 45 seconds. Bar - 100 urn. agent, has no effect on the larval arms. 4.3 Electrical stimulation The effects of A23187 can be reversed if the arms are rinsed and put into sea water Individual larvae that were competent to within 15 min. of treatment with the iono- metamorphose were stimulated with elec- phore, whereas treatment with X537A is not trical pulses of 6 to 15 Volts D.C. and reversible. If either ionophore is used 1 msec to 1 sec in duration. Stimuli were in conjunction with 10'2M EDTA or 10'^M administered singly or repetitively for 10 lanthanum nitrate contraction does not to 15 sec at a rate of 5 to 10 sec-1. If occur. the electrode was attached to any region Arms excised from competent larvae that of the ciliary band, epaulettes, or general are put into a small volume (50 ul) of sea body epidermis, each stimulus would produce water in which five larval arms have under- immediate and coordinated reversals of the gone the contraction and histolysis sequence will also undergo histolysis. After 15 minutes cilia cease beating and the cells Table 2. Effects of neurotransmitter sub- dissociate. There is neither apparent re- stances on isolated larval arms of Arbacia traction of cilia nor contraction of the punctulata. Two trials of 5 arms per larval epidermis prior to dissociation. treatment; + indicates contraction and Excised arms were also exposed to various histolysis resulted. neurotransmitter substances for periods of up to several hours (Table 2). Arms 10~3M 10_1»M 10"5M treated with 10_3, 10 ^ H noradrenaline, H "3 10 10 M dopamine, or 10 10 Acetylcholine chloride - 5 10~ M adrenaline undergo a sequence of Acetylthiocholine contraction and histolysis. The response iodide followed a time course similar to that Acetylcholine iodide - occurring during metamorphosis of intact Succinylcholine larvae, though the arms would not begin to chloride contract until 60 to 120 minutes after y-Aminobutyric acid treatment. Cells with meshworks of 5 to (gaba) 7 run filaments and retracted cilia and 5-Hydroxytryptamine pyknotic nuclei, were observed in arms that (serotonin) had been treated with catecholamines and Norepinephrine begun contraction. If whole, competent (noradrenaline) larvae are treated with catecholamines the L-epinephrine contraction and histolysis of larval epi- (adrenaline) dermis can be induced, though the adult 3-hydroxytyrosine rudiment would not become activated, nor (dopamine) would it be everted.

516 direction of ciliary beat, and twitch traction of the epidermis throughout its responses of muscles. In the case of length, but apparently additional forces Arbacia punctulata, an activation of the are involved in causing involution of the adult rudiment as indicated by movements of ciliary bands. the spines and tube feet would occur after Chia and Burke (1978) reported that during 5 to 10 stimuli of 100 msec each. For metamorphosis the larval epidermis of either species if the electrode is attached Dendraster excentricus becomes necrotic and to the oral surface of the adult rudiment, is phagocytized by cells of the larval gut. the larva responds to repeated stimulation The observations reported here support this by undergoing metamorphosis. In some cases idea and also show that mesenchyme is as few as 2 stimuli of 100 msec, or 20 involved in the phagocytosis of the histo- 1 msec stimuli delivered in 5 sec would lysized and necrotic tissues of the larval result in metamorphosis. Dendraster arms. Apparently the epidermis of the excentricus could also be induced to meta- larval arms first contracts and about two morphose by attaching the electrode to a minutes later histolysis and necrosis begin. region of the oral hood of the larva between The calcium ionophores A23187 and X537A the pre-oral and anterolateral arms (Fig. are a means of elevating the intracellular 10). Metamorphosis begins within the first calcium concentration of cells and have minute after stimulation, and follows the been used in a variety of biological systems usual sequence of metamorphic events. (Pressman, 1976). It has also been shown Juveniles appear normal and live indefin- that elevated levels of intracellular cal- itely. cium are associated with contracting non- The two regions that when stimulated would muscle cells (Lee and Auersperg, 1980) and elicit metamorphosis are associated with it has been suggested that an influx of dense accumulations of axons. The adult calcium may be required to initiate con- nerve ring is located in the oral surface traction (Hitchcock, 1977). The experiments of the adult rudiment, lying immediately reported here provide indirect evidence that beneath the 5 to 10 urn thick oral epi- such a hypothesis is correct. The calcium thelium, and a plexus of several hundred ionophores were able to induce contraction axons lies beneath the 3 to 5pm thick of the larval arms except when used in larval epidermis on the preoral hood (Burke, conjunction with a calcium chelating agent 1978). (EDTA) or an inhibitor of calcium flux (lanthanum) (Weiss, 1974). The ability of larval arms undergoing 5 DISCUSSION histolysis to induce dissociation of excised larval arms that have not been Cameron and Hinegardner (1978) observed stimulated to undergo contraction and histo- bands of dense fibrous material in the lysis suggests that there is some substance apical regions of cells of the larval epi- released from the histolysizing arms that thelium of Lytechinus pictus during meta- causes histolysis. Although further work morphosis, and report that the retraction is required to substantiate such a claim of the larval epithelium is reversibly it is notable that contraction and histo- inhibited with cytochalasin B. They sug- lysis can occur independently of each other. gest that the retraction of the larval epi- These experiments suggest that the develop- thelium may be facilitated by autonomously mental processes of contraction and histo- controlled changes in the shape of the cells lysis both lie innately programmed within of the larval epidermis. Several obser- the tissues of the larval arms, and during vations reported here for D. excentricus metamorphosis they are elicited in such a and A. punctulata support such a hypothesis, manner as to result in the retraction and within the epidermal cells, ciliary band resorption of the larval arms. cells, nerve cells, and mesenchymal cells, It has been previously reported that 5 to 7 nm filaments with the ultrastructural Arbacia punctulata larvae would metamorphose characteristics of actin were observed in response to electrical stimulation of during retraction of the larval arms. The 150 Volts delivered in pulses of 1 msec time of appearance and orientation of the (Cameron and Hinegardner, 1974). They did filaments are consistent with the hypothesis not note any specific locations for the that they produce the contractile forces placement of electrodes. The results responsible for the retraction of the larval reported here differ from those of Cameron arms. The ability of arms to contract and Hinegardner (1974) in that only very independently of other larval tissues low voltages were used, and subsequently, indicates that the contractile mechanism placement of the electrode was critical to resides within the arms. The retraction stimulating metamorphosis. Because the does not appear to simply involve a con- two locations used are intimately associated

517 with regions of neuropile it is presumed Hinegardner, R.T. 1969, Growth and develop- that these locations were effective because ment of the laboratory cultured sea direct stimulation of nervous tissues is urchin, Biol. Bull. mar. biol. Lab., necessary to elicit metamorohosi s . Woods Hole, 137: 465-475. These observations are consistent with the Hitchcock, S.E. 1977, Regulation of motility hypothesis that the nervous system mediates in non-muscle cells, J. Cell. Biol. 74: between perception of cues and the co- 1-15. O'dinated raetamorphic responses of the Hyman, L.H. 1955, The invertebrates:Echino- larval tissues. Presumably the electrical dermata, 763 pp. New York, McGraw-Hill. stimulation 01 neuropile activates nervous Lee, H.C. and N. Auersperg 1980, Calcium in elements which act either directly or in- epithelial cell contraction, J. Coll Biol. directly to initiate metamorphosis. Because 85: 325-336. catecho1amines appear to initiate develop- MacBride, E.W. 1903, The development of mental responses of metamorphosis, and have Echinus esculentus together with some been localized histochemically to the points on the development of E. miliaris axonal tracts of the ciliary bands (Ryberg, and E. acutus, Phil. Trans. R. Soc, Ser. 1974; Burke, unpublished observations), B. 195: 285-330. they may act as a chemical intermediary Pressman, B.C. 1976, Biological applications between nervous tissues and the tissues of of ionophores, Ann. Rev. Biochem. 45: 501- the larva which respond during metamor- 530. phosis. Ryberg, E. 1974, The localization of bio- genic amines in the echinopluteus, Acta Zool. 55: 179-189. 6 ACKNOWLEDGEMENTS Strathmann, M. 196SJ, Methods in development series. I. General procedures and This research was supported by a Smith- Echinodermata-echinoidea, Friday Harbor sonian Institution Post-doctoral Fellowship Laboratories, Friday Harbor, Washington, and a Natural Sciences and Engineering USA. Research Council of Canada University Strathmann, R.R. 1978, Larval settlement in Research Fellowship to the author. Dr. echinoderms. In F.5. Chia and M.E. Rice M.E. Rice graciously provided facilities (eds.), Settlement and metamorphosis of at the Ft. Pierce Bureau of the Smithsonian marine invertebrate larvae, p. 235-247. Insti tution. Weiss, G.B. 1974, Cellular pharmacoloov of lanthanum, Ann. Rev. Pharmacol. 14: 343- 354. 7 REFERENCES

Burke, R.D. 1978, The structure of the nervous system of the pluteus larva of Strongylocentrotus purpuratus, cell Tiss. Res. 191:233-247. Burke, R.D. 1980, Podial sensory receptors and the induction of metamorphosis in echinoids, J. exp. mar. Biol. Ecol. 47: 223-234. Cameron, R.A. and R.T Hinegardner 1974, Initiation oi metamorphosis in laboratory cultured sea urchins, Biol. Bull. mar. biol. Lab., Woods Hole, 146: 3.15-342. Cameron, R.A. and R.T. Hinegardner, 1978, Early events in metamorphosis, description and analysis, J. morph. 157: 21-32. Chia, F.S. and R.D. Burke 1978, Eehinoderm metamorphosis: fate of larval structures. In P.S. Chia and M.E. Rice (eds.), Settle- ment and metamorphosis of marine inverte- brate larvae, p. 219-234. New York, Elsevier. Highsmith, R. 1977, Larval substrate selection, metamorphosis, and mortality in the sand dollar Dendraster excentricus, Am. Zool. 17: 935.

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